Process for producing physiologically active protein using genetically modified silkworm

ABSTRACT

The present invention provides a genetic engineering material for insects that enables a target protein to be purified easily, without requiring the use of recombinant baculovirus, while simultaneously providing a process for producing exogenous protein using that genetic engineering material. A gene recombinant silkworm is obtained by inserting an exogenous protein gene such as a cytokine gene coupled to a promoter that functions in silk glands into a silkworm chromosome. An exogenous protein such as a cytokine is then extracted and purified from the silk glands or cocoon of that silkworm or its offspring. A large amount of exogenous protein can be produced within silk gland cells, outside silk gland cells or in silk thread or a cocoon by inserting an expression gene cassette, in which the DNA sequence of the 3′ terminal portion and the DNA sequence of the 5′ terminal portion of fibroin H chain gene are fused to the exogenous protein gene, into silk gland cells and so forth.

TECHNICAL FIELD

The present invention relates to a process for producing a recombinantcytokine using a silkworm incorporating a cytokine gene in itschromosomes. In addition, the present invention relates to a generecombinant silkworm having the property of producing a recombinantcytokine in a silk gland or cocoon and silk thread, and a vector forinserting an exogenous gene into silkworm chromosomes for producing therecombinant silkworm. In addition, the present invention also relates toa process for producing exogenous protein using insect cells, insecttissue or insects to which a gene has been inserted using theaforementioned vector. Moreover, the present invention relates to silkthread containing an exogenous protein produced by a recombinantsilkworm obtained in the present invention.

BACKGROUND ART

The production of exogenous proteins using gene recombination technologyis used in various industries. The hosts used for their productionconsist mainly of E. coli, yeast, animal cells, plant cells and insectcells. However, a host has yet to be developed that is capable ofefficiently producing all kinds of exogenous proteins and, as it isnecessary to construct a production system for each target protein, atechnical breakthrough is being sought in technology for producingexogenous proteins in individual hosts.

Systems using bacteria like E. coli or yeast have problems withposttranslational modification, and in some cases these systems areunable to synthesize proteins in a form that allows them to functionadequately. In addition, although animal cells allow proteins to besynthesized in a functional form, it is typically difficult to growthese cells and the production volume is low thereby making thisuneconomical.

On the other hand, in the production of gene recombinant proteins usinginsects or insect cells, useful proteins having enzymatic orphysiological activity can be produced comparatively inexpensively andmodifications can be obtained, following protein translation, thatresemble those in animals. More specifically, a method in which abaculovirus incorporated a recombinant exogenous gene is infected intoinsects or insect cells allows the exogenous protein to be producedcomparatively inexpensively, and physiologically active proteins areknown that have been commercialized as pharmaceuticals (JapaneseUnexamined Patent Publication Nos. 61-9288 and 61-9297).

In the case of the production of cytokines, which are physiologicallyactive substances having immunoregulatory functions and which areattracting attention in pharmaceutical applications, methods aredisclosed in Japanese Unexamined Patent Publication Nos. 3-139276 and9-234085 in which silkworms are inoculated with BmNPV containing afeline interferon-ω gene and a canine interferon-γ gene, respectively.In addition, a process for producing human collagen using insect cellsinfected with baculovirus is known as an example of the production of aprotein other than interferon using insects (Japanese Unexamined PatentPublication No. 8-23979).

However, as technologies for producing recombinant proteins usinginsects or insect cells of the prior art use a recombinant virus toincorporate an exogenous gene, there is the problem of the need fordeactivation or containment of recombinant virus from the viewpoint ofsafety. In addition, in methods in which a recombinant virus isinoculated into a silkworm, as the inoculation of the recombinant virusis troublesome task and the target exogenous protein is produced insilkworm hemolymph, it is necessary to purify the target recombinantprotein from the large amount of contaminating proteins originating inthe body fluids of the silkworm. Consequently, there was the problem ofit being difficult to obtain a highly pure recombinant protein.

On the other hand, attempts have been made in recent years to recombineexogenous genes into insect chromosomes, and a method has been developedthat uses homologous recombination to introduce and express in silkwormchromosomes a fused gene in which jellyfish green fluorescence-proteingene was coupled to silkworm fibroin L chain gene using DNA ofAutographa californica nuclear polyhedrosis virus (AcNPV), which is atype of nuclear polyhedrosis virus (Genes Dev., 13, 511-516, 1999), anda silkworm containing human collagen gene and a production process havebeen disclosed that utilize this technology (Japanese Unexamined PatentPublication No. 2001-161214). Recently, research has been conducted on amethod for expressing a protein encoded by an exogenous gene by stablyintroducing that exogenous gene into silkworm chromosomes usingpiggyBac, which is a transposon originating in a lepidopteron, using thejellyfish green fluorescence protein as a model, and the gene has beenconfirmed to be stably propagated to offspring by mating (NatureBiotechnology, 18, 81-84, 2000).

However, in the aforementioned method for inserting an exogenous proteingene into insect chromosomes using AcNPV, as a recombinant baculovirus(AcNPV) is used, there is still the problem of having to deactivate andcontain the recombinant virus. In the example that used the piggyBactransposon, as the amount of green fluorescence protein produced isinadequate and as it is also produced throughout the silkworm,sophisticated purification technology is required to recover theexpressed recombinant green fluorescence protein in a highly pure form,thereby resulting in the method being uneconomical. In addition, theamount of recombinant protein produced is inadequate and extremely low.

Namely, in this technology for producing an exogenous protein usinginsect cells as a host, there are several problems such as the need todeactivate and contain the recombinant baculovirus, the difficulty inpurifying the target protein from body fluid in which a large amount ofcontaminating proteins are present, as in the case of using silkworms,and the expressed amount of the target protein being low.

There are no known examples, thus far, of expressing a target protein byinserting a gene that encodes a physiologically active protein such as acytokine gene into silkworm chromosomes. In addition, there are also noexamples of having recovered a recombinant cytokine from a site, otherthan silkworm body fluid, such as a silk gland or silk thread secretedby silkworms, and confirming the physiological activity of the resultingcytokine. In addition, there are also no precedents regarding a silkwormcapable of inheriting such properties. In addition, there are noexamples of having produced a large amount of recombinant protein insilk thread using a recombinant silkworm produced using a transposon.

DISCLOSURE OF THE INVENTION

Although extensive research has been conducted on technologies forproducing recombinant proteins using insects, there are problems such asthe need to deactivate and contain the recombinant baculovirus in whichthe exogenous protein gene has been incorporated, or the need to take alot of time and labor associated with inoculating the recombinant virus.In addition, the production of exogenous protein in silkworms usingrecombinant baculovirus had the problem of it being difficult to extractand purify the target protein from body fluid containing large amountsof contaminating proteins.

Although studies have been conducted on technologies, for producingrecombinant proteins, in which an exogenous protein gene has beeninserted into silkworm chromosomes, these have problems consisting ofthe small amount of target exogenous protein produced and the difficultyin purifying the target protein from silkworm body fluid.

In consideration of these circumstances, the object of the presentinvention is to provide a genetic engineering material for insects thatdoes not require the use of recombinant baculovirus and enables a targetprotein having physiological activity to be purified easily, whilesimultaneously providing a process for producing exogenous protein usingthat genetic engineering material.

As a result of extensive studies, the inventors of the present inventionfound that, by inserting a DNA sequence having a structure in which agene that encodes a target protein is coupled downstream from a promoterspecifically expressed in silkworm silk glands into silkworm chromosomesusing DNA originating in a transposon, the target protein is produced inthe silk glands, or the cocoon and the silk thread, in a form thatretains physiological activity, thereby leading to completion of thepresent invention. In the present invention, as the recombinant proteincan be recovered from the silk glands or silk and cocoon thread withoutcontaining a large amount of contaminants, it offers the advantage ofallowing the target protein to be purified easily. Moreover, as a viruslike baculovirus is not used, virus deactivation is not necessarythereby allowing the recombinant protein to be produced both easily andsafely.

In addition, as a result of conducting extensive studies focusing on thefact that the silkworm silk glands, and particularly the posterior silkgland, produces a large amount of fibroin that accounts for 70 to 80% ofsilk protein, and that the fibroin is secreted by the silk gland cells,the inventors of the present invention found that the amount ofexogenous protein produced is increased considerably by inserting intosilk gland cells a gene cassette in which the 5′ terminal of anexogenous protein gene is coupled to the 3′ terminal of a fibroin Hchain gene 5′ terminal portion containing a first intron of fibroin Hchain gene downstream from a promoter expressed in the silk glands sothat the amino acid frames are continuous. In addition, it was alsofound that a large amount of exogenous protein is secreted and producedby silk gland cells when a fused gene in which the 3′ terminal portionof fibroin H chain gene is coupled to the 3′ side of an exogenousprotein gene, so that the amino acid frames are continuous, is expressedunder the control of a promoter expressed in silk glands. In addition,it was also found that a recombinant silkworm produces a large amount ofa target protein in its silk threads when a gene cassette was producedin which a DNA sequence of the 5′ terminal portion containing a firstintron of fibroin H chain gene on the 5′ side of an exogenous proteingene, and a DNA sequence of the 3′ terminal portion of fibroin H chaingene on the 3′ side, were respectively designed so that their amino acidframes were continuous, followed by producing a recombinant silkworm inwhich that gene cassette was inserted into its chromosomes.

The inventors of the present invention succeeded in producing a largeamount of exogenous protein in silk gland cells, outside silk glandcells and in silk thread by inserting into silk gland cells and so forthan expression gene cassette in which the DNA sequence of the 5′ terminalportion and the DNA sequence of the 3′-portion of fibroin H chain genewere fused to an exogenous protein gene, and were able to establish anexogenous protein-production technology that facilitates purification byproducing an exogenous protein using silk glands instead of using arecombinant baculovirus.

Namely, the present invention relates to a process for producing arecombinant cytokine comprising producing a gene recombinant silkwormthat incorporates a cytokine gene in its chromosomes, and recovering thecytokine from the silk glands or cocoon and silk thread. Moreover, thepresent invention also relates to a gene recombinant silkworm in which acytokine gene is incorporated, and a gene recombinant vector used toinsert the cytokine gene into the silkworm.

Moreover, the present invention relates to a genetic engineeringmaterial, such as the gene cassette or vector described below, capableof being used for exogenous protein production in insects, atransformant, a process for producing exogenous protein using thattransformant, and silk thread containing exogenous protein.

Thus, the present invention provides 1) a gene cassette for expressingan exogenous protein comprising (1) a promoter expressed in silk glands,and (2) a gene coupled downstream from (1) in which the 5′ terminalportion of fibroin H chain gene is fused to the 5′ side of an exogenousprotein-structural gene.

Moreover, the present invention provides 2) a gene cassette forexpressing an exogenous protein comprising (1) a promoter expressed insilk glands, and (2) a gene coupled downstream from (1) in which the 3′terminal portion of fibroin H chain gene is fused to the 3′ side of anexogenous protein structural gene not containing a stop codon.Alternatively, the present invention provides a gene cassette forexpressing an exogenous protein comprising (1) a promoter expressed insilk glands, and (2) a gene coupled downstream from (1) in which anexogenous protein structural gene is fused to the 3′ side of the 3′terminal portion of fibroin H chain gene.

Moreover, the present invention provides 3) a gene cassette forexpressing an exogenous protein comprising (1) a promoter expressed insilk glands, and (2) a gene coupled downstream from (1) in which the 5′terminal portion of fibroin H chain gene is fused to the 5′ side of anexogenous protein structural gene not containing a stop codon, and the3′ terminal portion of fibroin H chain gene is fused to the 3′ side ofthe structural gene.

In addition, the present invention provides 4) an expression vector forinsect cells containing a gene cassette for expressing an exogenousprotein according to any of the aforementioned 1) through 3).

Moreover, the present invention provides 5) a process for producingexogenous protein comprising inserting an expression vector for insectcells according to the aforementioned 4) into insect cells.

Moreover, the present invention provides 6) a process for producingexogenous protein comprising producing a recombinant silkworm in which agene cassette for expressing an exogenous protein according to any ofthe aforementioned 1) through 3) is incorporated in its chromosomes, andafter producing the exogenous protein in the silk glands or silk threadof the resulting recombinant silkworm, recovering the exogenous proteinfrom the silk glands or silk thread.

In addition, the present invention provides 7) a recombinant silkworm inwhich a gene cassette for expressing an exogenous protein according toany of the aforementioned 1) through 3) is incorporated in itschromosomes, and has the ability to produce exogenous protein in itssilk glands or silk thread.

Moreover, the present invention provides 8) a silk thread containing anexogenous protein produced by the silkworm according to theaforementioned 7).

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a drawing showing a restriction map of gene insertion vectorpigSIB.

FIG. 2 is a drawing showing a restriction map of gene insertion vectorpigFIB.

FIG. 3 is a drawing showing a restriction map of plasmid pHA3PIG havinga transposase.

FIG. 4 is a drawing showing the results of treating the genomic DNA of11 silkworms (G1) obtained from the positive moth groups of Table 1 withEcoRV and XmnI, followed by performing Southern blotting analysis usinga feline interferon-ω gene as a probe.

FIG. 5 is a drawing showing the antiviral activity of a silk threadextract of a recombinant silkworm into which was inserted felineinterferon-ω gene coupled to fibroin H chain promoter. The sample in thedyed lane is shown to have activity.

FIG. 6 is a drawing showing the results of treating silkworm silk glandgenomic DNA obtained from the positive moth groups of Table 3 (3 mothgroups from Experiment 1 and 2 moth groups from Experiment 2) with EcoRIor Bg1II followed by performing Southern blotting analysis using felineinterferon-w gene as a probe.

FIG. 7 is a drawing of the detection of expression of feline interferonmRNA in a middle silk thread of a gene recombinant silkworm by RT-PCR.

FIG. 8 is a drawing showing the antiviral activity of a middle silkgland extract and a cocoon and silk thread extract of a recombinantsilkworm into which was inserted feline interferon-w gene coupled to asericin promoter. The sample in the dyed lane is shown to have activity.

FIG. 9 is a drawing showing the procedure for producing a construct forgene insertion containing a P-IC-A gene cassette (first half).

FIG. 10 is a drawing showing the procedure for producing a construct forgene insertion containing a P-IC-A gene cassette (second half).

FIG. 11 is a drawing showing the procedure for producing a construct forgene insertion containing an HP-IC-HA gene cassette (first half).

FIG. 12 is a drawing showing the procedure for producing a construct forgene insertion containing an HP-IC-HA gene cassette (second half).

FIG. 13 is a drawing showing the procedure for producing a construct forgene insertion containing an HUP-IC-HA gene cassette (first half).

FIG. 14 is a drawing showing the procedure for producing a construct forgene insertion containing an HUP-IC-HA gene cassette (second half).

FIG. 15 is a drawing showing the procedure for producing a construct forgene insertion containing an HP-IC-A gene cassette (first half).

FIG. 16 is a drawing showing the procedure for producing a construct forgene insertion containing an HP-IC-A gene cassette (second half).

FIG. 17 is a drawing of the analysis of the expression ofβ-galactosidase in cultured silkworm silk glands by western analysis.The first exon, first intron and second exon regions of fibroin H chaingene were clearly determined to play an important role in synthesis orgene expression of proteins within cells. In addition, secretion outsidethe cells was also confirmed.

FIG. 18 is a drawing of the analysis of the expression of recombinantprotein in silkworm silk gland tissue by Western analysis. The firstexon, first intron and second exon regions of fibroin H chain gene werereconfirmed to play an important role in dramatically improving theexpression of recombinant protein in silkworm posterior silk glandcells. In addition, a gene region that improves the amount of proteinproduced was found in a roughly 5.5 kbp upstream region from the fibroinpromoter.

FIG. 19 is a drawing of the analysis of the production of recombinantprotein in silk thread by western analysis. The 3′ terminal portion offibroin H chain gene was determined to play an important role insecretion of protein synthesized within silk gland cells to silk thread.In addition, a gene region that improves the amount of protein producedwas reconfirmed in the roughly 5.5 kbp upstream region from the fibroinpromoter.

BEST MODE FOR CARRYING OUT THE INVENTION

Cytokines are proteins produced by various cells that haveimmunoregulatory activity, antiviral activity and blood cell growthactivity on hematopoietic cells and immunocytes. Their activity isdemonstrated as a result of forming a precise higher order structure andbonding to specific receptors on the cell membrane. They have previouslybeen applied clinically to humans and animals based on thecharacteristics of their activity.

Although there are no particular limitations on the cytokines of thepresent invention, they should be cytokines for which theirphysiological activity is maintained when expressed in silkworms,examples of which include physiologically active substances havingimmunoregulatory activity, antiviral activity or blood cell growthactivity and so forth such as human interferon-α, β and γ (J. InterferonRes. 5, 521-526, 1985; Nucleic Acids Res. 10, 2487-2501, 1982), humaninterleukin-12 (J. Immunol. 146, 3074-3081; 1991), human granulocytecolony stimulating factor (Nature, 319, 415-418, 1986), humanerythropoietin (Nature, 313, 806-810, -1985), human thrombopoietin(Cell, 77, 1177-1124, 1994), feline interferon-ω, (Biosci. Biotech.Biochem., 56, 211-214, 1992, GenBank database registration no. E04599),feline erythropoietin (GenBank database registration no. FDU00685),feline granulocyte colony stimulating factor (Gene, 274, 263-269, 2001),canine interferon-γ (GenBank database registration no. S41201), canineinterleukin-12 (Japanese Unexamined Patent Publication No. 10-36397) andcanine granulocyte colony stimulating factor (U.S. Pat. No. 5,606,024).Preferable examples of cytokines include interferons and colonystimulating factors, and these preferably include interferon-α, β, γ, ωand τ along with colony stimulating factor, erythropoietin andthrombopoietin. More preferable examples of cytokines include felineinterferon-ω, feline granulocyte colony stimulating factor and humaninterferon-β.

Feline interferon-ω gene is obtained by cutting out from a plasmidextracted from, for example, E. coli (pFeIFN1) (Patent MicroorganismDepository No. 1633). In addition, this gene can also be obtained fromrBNV100 produced by co-transfecting into established silkworm cells witha recombinant plasmid produced by ligating feline interferon-ω gene to asilkworm cloning vector (T. Horiuchi, et al., Agric. Biol. Chem., 51,1573-1580, 1987), and silkworm nuclear polyhedrosis virus.

Feline granulocyte colony stimulating factor can be obtained bystimulating CRFK cells, which are cultured cells originating in felinekidney, with LPS followed by recovering mRNA from the cells and thencarrying out PCR using the cDNA obtained by reverse transcription as atemplate and using primers established with reference to GenBankdatabase registration no. AB042552.

Human interferon-β gene can be acquired by cutting out from plasmidORF-hIFN-β (Invitrogen) that encodes its cDNA.

The method for inserting a gene into silkworm chromosomes used in thepresent invention should enable the gene to be stably incorporated andexpressed in the chromosomes, and be stably propagated to offspring, aswell, by mating. Although a method using micro-injection into silkwormeggs or a method using a gene gun can be used, a method that is usedpreferably consists of the micro-injection into silkworm eggs with atarget gene containing vector for insertion of an exogenous gene intosilkworm chromosomes and helper plasmid containing a transposon gene(Nature Biotechnology 18, 81-84, 2000) simultaneously.

The target gene is inserted into reproductive cells in a recombinantsilkworm that has been hatched and grown from the micro-injectedsilkworm eggs. Offspring of a recombinant silkworm obtained in thismanner are able to stably retain the target gene in their chromosomes.The gene recombinant silkworm obtained in the present invention can bemaintained in the same manner as ordinary silkworms. Namely, up to fifthinstar silkworms can be raised by incubating the eggs under normalconditions, collecting the hatched larva to artificial feed and thenraising under the same conditions as ordinary silkworms.

Gene recombinant silkworms obtained in the present invention are able topupate and produce a cocoon in the same manner as ordinary silkworms.Males and females are distinguished in the pupa stage, and after havingtransformed into moths, males and females mate and eggs are gathered onthe following day. The eggs can be stored in the same manner as ordinarysilkworm eggs. The gene recombinant silkworms of the present inventioncan be maintained on subsequent generations by repeating the breeding asdescribed above, and can be increased to large numbers.

The exogenous gene insertion vector used for the purpose of inserting acytokine gene used in the present invention into silkworm chromosomes isnot subject to any particular limitations provided it is designed so asto precisely control cytokine expression. Normally, it has a structurein which the cytokine gene is coupled to downstream from a promoterspecifically expressed in the silk glands and upstream from an arbitrarypoly A sequence, and has a pair of DNA sequences originating in atransposon outside these gene sequences. Moreover, a signal sequenceoriginating in an arbitrary gene may be coupled between the cytokinegene and the promoter, and an arbitrary gene sequence may also becoupled between the cytokine gene and poly A. In addition, anartificially designed and synthesized gene sequence can also be coupled.In addition, a sequence for replication within a bacterial host,antibiotic resistance gene, fluorescence protein gene or LacZ gene andso forth can also be coupled as necessary. For example, the gene ofgreen fluorescence protein GFP coupled downstream from a suitablepromoter can be inserted at a suitable location between a pair oftransposon DNA sequences. As a result, this facilitates screening forgene recombinant silkworms. In addition, this vector may also containall or a portion of pUC9, pUC19 or other plasmids originating in E.coli.

Moreover, although there are no particular limitations on the promoterused here, and any promoter originating in any organism can be usedprovided its acts effectively within silkworm cells, a promoter that hasbeen designed to specifically induce protein in silkworm silk glands ispreferable. Examples of silkworm silk gland protein promoters includefibroin H chain promoter, fibroin L chain promoter, p25 promoter andsericin promoter.

Examples of other gene sequences used in addition to the promoterinclude signal sequences, poly A sequences and other sequences thatcontrol gene expression. These are not limited to specific genesequences, but rather those which are suitable for expression of thetarget gene can be selected. Examples include sequences originating inthe target protein such as signal sequences of cytokines such as felineinterferon-o and poly A sequences, and signal sequences and poly Asequences of insect protein contained in the silkworm serving as thehost. Alternatively, other examples include sequences that have beenproven to be generally effective for expressing proteins such as SV40poly A and bovine growth hormone poly A. By changing the gene sequenceof the aforementioned promoters and the other sequences coupled with thecytokine genes, the locations where they are expressed and the amountsexpressed can be controlled.

In the present invention, a “gene cassette for expressing an exogenousprotein” refers to a set of DNA required for a synthesis of proteinencoded by the exogenous protein structural gene in the case of beinginserted into insect cells. This gene cassette for expressing anexogenous protein contains an exogenous protein structural gene and apromoter that promotes expression of that gene. Normally, it alsocontains a terminator and poly A addition region, and preferablycontains a promoter, exogenous protein structural gene, terminator andpoly A addition region. Moreover, it may also contain a secretion signalgene coupled between the promoter and the exogenous protein structuralgene. An arbitrary gene sequence may also be coupled between the poly Aaddition sequence and the exogenous protein structural gene. Inaddition, an artificially designed and synthesized gene sequence canalso be coupled.

In addition, a “gene cassette for inserting a gene” refers to a genecassette for expressing an exogenous gene having an inverted repetitivesequence of a pair of piggyBac transposons on both sides, and consistingof a set of DNA inserted into insect cell chromosomes through the actionof the piggyBac transposons.

There are no particular limitations on the method used to acquire DNAused in the present invention. Examples of such methods include a methodin which a required gene region is amplified and acquired using apolymerase chain reaction (PCR) based on known genetic information, anda method in which a genome library or cDNA library is screened usinghomology as an indicator based on known genetic information. In thepresent invention, these genes include variants resulting from geneticpolymorphism and artificial mutation treatment using mutagens and soforth. Genetic polymorphism refers to that in which a portion of thebase sequence of a gene is altered by a sudden spontaneous mutation inthe gene.

Although there are no particular limitations on the promoter in the genecassette for expressing an exogenous protein, that having a high levelof activity that promotes expression of an exogenous protein gene ispreferable. Although examples include the promoter of drosophila heatshock protein gene described in Japanese unexamined Patent PublicationNo. 6-261770 and the promoter of silkworm actin gene (NatureBiotechnology 18, 81-84, 2000), promoters having a high level ofpromoting activity in silkworm silk gland cells are preferable, examplesof which include the promoters of fibroin H chain gene (base numbers255-574 of GenBank registration no. V00094), fibroin L chain gene (Gene,100, 151-158; GenBank registration no. M76430) and sericin gene (basenumbers 599-1656 of GenBank registration no. AB007831).

“Exogenous protein structural gene” refers to a gene not possessed byhost cells in which a gene is to be expressed, and which encodes aprotein not inherently produced by the host cells. Although there are noparticular limitations thereon, in consideration of industrial value,examples include genes of proteins that are produced by humans ormammals such as genes of growth hormones, cytokines, growth factors andcell structural proteins. In addition, genes of enzymes and variousproteins produced by microbes, plants or insects are also included inthe scope of the present invention.

In the gene cassette for expressing an exogenous protein in the presentinvention, the 5′ terminal portion of fibroin H chain gene is a DNAsequence having action that enhances expression of exogenous proteingene by a promoter, and contains a first exon of fibroin H chain gene,all or a portion of a first intron, and a portion of a second exon. Byfusing the 51 side of an exogenous protein structural gene to the 3′side of this second exon so that the amino acid reading frame iscontiguous, the amount of exogenous protein produced can be improved.However, since surplus amino acid residues are added to the N terminalside of the target exogenous protein if the second axon portion is toolong, there are cases in which the structure or activity of the targetexogenous protein is lost. Consequently, it is necessary that the secondexon portion have a suitable length according to the purpose. In manycases, favorable results can be obtained by making the second exonportion to extend to immediately after or up to several amino acidresidues from the secretion signal gene of fibroin H chain gene. Inaddition, as the region upstream from the 5′ side of fibroin H chaingene promoter, namely a roughly 5.5 kbp upstream region, is consideredto be the region that enhances promoter activity, adding this region canbe expected to increase the amount of target protein expressed.

In the case of producing an exogenous protein in silkworm silk glands,the 3′ terminal portion of fibroin H chain gene is a DNA sequence havingthe effect of causing secretion of a large amount of exogenous proteinoutside the silk gland cells. A recombinant silkworm in which a genecassette for expressing an exogenous protein, in which the 3′ terminalportion of fibroin H chain gene serving as the signal for secreting intosilk thread is fused to the 3′ side, is inserted into its chromosomes isable to produce exogenous protein in its silk thread. In addition, the3′ terminal portion of fibroin H chain gene may be present upstream ordownstream from the exogenous protein gene or within the exogenousprotein gene.

In the case at least one cysteine residue is present in this portion andthe 3′ terminal of fibroin H chain gene is used as is, the cysteineresidue is located at the 20th residue from the carboxyl terminal of thefibroin H chain gene. This cysteine fulfills the role of bonding tofibroin L chain by a disulfide bond. There are no particular limitationson the length of the DNA sequence of the 3′ terminal portion of fibroinH chain gene provided it does not inhibit formation of the disulfidebond with fibroin L chain. As a repetitive DNA sequence continues fromabout 100 or more bases upstream from the 3′ terminal of fibroin Hchain, cleaving the DNA sequence of this upstream portion to anarbitrary length is difficult with a restriction endonuclease. Thus, inconsideration of the ease of genetic engineering techniques, roughly 100base pairs on the 3′ portion where the repetitive DNA sequence offibroin H chain gene ends can be preferably used for the 3′ terminalportion of fibroin H chain. In addition, as a large number of aminoacids originating in the carboxyl terminal of fibroin H chain proteinbond to the carboxyl terminal or amino terminal of the exogenous proteinif the 3′ terminal portion of fibroin H chain gene is excessively long,there are cases in which the structure or activity of the targetexogenous protein is lost. Thus, there are cases in which it isnecessary to make the DNA sequence of the 3′ terminal portion of fibroinH chain gene as short as possible depending on the target protein.

Although there are no particular limitations on the poly A region, apoly A region of a protein gene expressed in large amounts in silkglands, such as fibroin H chain, fibroin L chain or sericin, can be usedpreferably.

A vector in the present invention refers to that having a cyclic orlinear DNA structure. A vector capable of replicating in E. Coli andhaving a cyclic DNA structure is particularly preferable. This vectorcan also incorporate a marker gene such as an antibiotic resistance geneor jellyfish green fluorescence protein gene for the purpose offacilitating selection of transformants.

Although there are no particular limitations on the insect cells used inthe present invention, they are preferably lepidopteron cells, morepreferably Bombyx mori cells, and even more preferably silkworm silkgland cells or cells contained in Bombyx mori eggs. In the case of silkgland cells, posterior silk gland cells of fifth instar silkworm larvaare preferable because there is active synthesis of fibroin protein andthey are easily handled.

There are no particular limitations on the method used to incorporate agene cassette for expression of exogenous protein and a vector into theinsect cells. Although the calcium phosphate method, methods usingelectroporation, methods using liposomes, methods using a gene gun andmethods using micro-injection can be used for incorporation intocultured insect cells, in the case of incorporating into silkworm silkgland cells, for example, a gene can be easily incorporated intoposterior silk gland tissue removed from the body of a fifth instarsilkworm larvae using a gene gun.

Gene incorporation into the posterior silk gland using a gene gun can becarried out by, for example, bombarding gold particles coated with avector containing a gene cassette for expressing exogenous protein intoa posterior silk gland immobilized on an agar plate and so forth using aparticle gun (Bio-Rad, Model No. PDS-1000/He) at an He gas pressure of1,100 to 1,800 psi.

In the case of incorporating a gene into cells contained in eggs ofBombyx mori, a method using micro-injection is preferable. Here, in thecase of performing micro-injection into eggs, it is not necessary tomicro-inject into the cells of the eggs directly, but rather a gene canbe incorporated by simply micro-injecting into the eggs.

A recombinant silkworm containing the “gene cassette for expressing anexogenous protein” of the present invention in its chromosomes can beacquired by micro-injecting a vector having a “gene cassette forinserting a gene” into the eggs of Bombyx mori. For example, a firstgeneration (G1) silkworm is obtained by simultaneously micro-injecting avector having a “gene cassette for inserting a gene” and a plasmid inwhich a piggyBac transposase gene is arranged under the control ofsilkworm actin promoter into Bombyx mori eggs according to the method ofTamara, et al. (Nature Biotechnology 18, 81-84, 2000), followed bybreeding the hatched larva and crossing the resulting adult insects (G0)within the same group. Recombinant silkworms normally appear at afrequency of 1 to 2% among this G1 generation.

Selection of recombinant silkworms can be carried by PCR using primersdesigned based on the exogenous protein gene sequence after isolatingDNA from the G1 generation silkworm tissue. Alternatively, recombinantsilkworms can be easily selected by inserting a gene encoding greenfluorescence protein coupled downstream from a promoter capable of beingexpressed in silkworm cells into a “gene cassette for inserting a gene”in advance, and then selecting those individuals that emit greenfluorescence under ultraviolet light among G1 generation silkworms atfirst instar stage.

In addition, in the case of the micro-injection of a vector having a“gene cassette for inserting a gene” into Bombyx mori eggs for thepurpose of acquiring recombinant silkworms containing a “gene cassettefor expressing an exogenous protein” in their chromosomes, recombinantsilkworms can be acquired in the same manner as described above bysimultaneously micro-injecting a piggyBac transposase protein.

A piggyBac transposon refers to a transfer factor of DNA having aninverted sequences of 13 base pairs on both ends and an ORF inside ofabout 2.1 k base pairs. Although there are no particular limitations onthe piggyBac transposon used in the present invention, examples of thosethat can be used include those originating in Trichoplusia ni cell lineTN-368, Autographa californica NPV (AcNPV) and Galleria mellonea NPV(GmMNPV). A piggyBac transposon having gene and DNA transfer activitycan be preferably prepared using plasmids pHA3PIG and pPIGA3GFP having aportion of a piggyBac originating in Trichoplusia ni cell line TN-368(Nature Biotechnology 18, 81-84, 2000).

The structure of the DNA sequence originating in a piggyBac is requiredto have a pair of inverted terminal sequences containing a TTAAsequence, and has an exogenous gene such as a cytokine gene insertedbetween those DNA sequences. It is more preferable to use a transposasein order to insert an exogenous gene into silkworm chromosomes using aDNA sequence originating in a transposon. For example, the frequency atwhich a gene is inserted into silkworm chromosomes can be improvedconsiderably by simultaneously inserting DNA capable of expressing apiggyBac transposase to enable the transposase transcribed andtranslated in the silkworm cells to recognize the two pairs of invertedterminal sequences, cut out the gene fragment between them, and transferit to silkworm chromosomes.

The gene recombinant silkworm used in the present invention refers to asilkworm which has had inserted into its chromosomes an exogenousprotein gene, and after treating the silkworm chromosomal DNA withrestriction endonuclease in accordance with ordinary methods, yields apositive signal when subjected to Southern blotting using the exogenousprotein gene labeled in accordance with ordinary methods as a probe.There are no particular limitations on the gene locus on the chromosomeinto which a cytokine gene has been inserted provided it is a site thatdoes not inhibit silkworm development, differentiation and growth. Therecombinant silkworm has the ability to produce exogenous protein in itssilk gland cells, silk gland lumen and silk thread. In addition, therecombinant silkworm is able to develop and mate normally, stably retainthe inserted exogenous protein gene, and transmit that gene to itsoffspring. Thus, the amount of exogenous protein produced can easily beincreased by increasing the number of recombinant silkworms throughcrossing. Crossing between transgenic silkworm strain and non-transgenicstrain can increase the amount of the produced exogenous protein. Inthis case, it is necessary to cross the silkworms while suitablyselecting those silkworms into which the target exogenous protein genehas been inserted. In this case, offspring that have inherited the geneof the recombinant silkworm can be easily evaluated by analyzing amarker gene used to select the recombinant silkworms or the presence orstructure of the exogenous protein gene by PCR or Southern blotting andso forth using cell DNA obtained from an arbitrary tissue.

Insect cells and silkworm silk glands containing the gene cassette forexpression of an exogenous protein of the present invention can produceexogenous protein in culture supernatant or their cells by respectivelyculturing in culture liquid suitable for their culturing. For example,BmN cells, which are silkworm ovary cells that have been inserted withthe expression gene cassette of the present invention, produce a targetexogenous protein after 3 or 4 days of culturing by culturing at 27° C.in TC-100 medium (PharMingen). In addition, silkworm posterior silkgland produces exogenous protein by culturing at 25° C. in Grace'sinsect medium after being excised aseptically from, for example,fifth-instar larva. In the case of producing protein in silk glands, itis preferable to maintain a high dissolved oxygen concentration in themedium, and culture while removing low molecular weight factors thatinhibit protein synthesis that accumulate in the medium by, for example,an ultrafiltration membrane since this allows protein synthesis toproceed for a long period of time.

A silk gland inserted with an exogenous protein gene fused to the 3°terminal of fibroin H chain gene of the present invention is capable ofproducing a large amount of a target exogenous protein in culturesupernatant. Since nearly all contaminating proteins in the silk glandculture supernatant are fibroin, the target protein can be easilypurified from the silk gland culture supernatant, and as a result, ahighly pure target protein can be obtained.

The recombinant silkworm obtained in the present invention can be raisedin the same manner as ordinary silkworms, and is able to produceexogenous protein by raising under ordinary conditions. The amount ofexogenous protein produced can be improved by optimizing thetemperature, humidity and feeding conditions, etc. during the fifthinstar period in particular corresponding to the target exogenousprotein.

A recombinant silkworm inserted with an exogenous protein gene fused tothe 3′ terminal of fibroin H chain gene of the present invention is ableto produce a large amount of a target exogenous protein in its cocoon.The target exogenous protein can also be easily purified and recoveredfrom the resulting cocoon. In addition, depending on the function of theexogenous protein produced, silk thread containing the resultingexogenous protein can be used directly or in a partially processed formin various industrial applications.

Exogenous protein can be obtained from the silk gland or cocoon and silkthread of a recombinant silkworm obtained in the present invention by asuitable extraction procedure. Although there are no particularlimitations on the solvent used to extract exogenous protein from silkglands or cocoon and silk thread, an aqueous solvent system ispreferable in many cases. An aqueous solution used for extraction maycontain a suitable solute for promoting extraction of the exogenousprotein, examples of which include inorganic acids such as phosphoricacid, organic acids such as acetic acid, citric acid and malic acid,salts such as sodium, chloride, urea, guanidine hydrochloride andcalcium chloride, and polar organic solvents such as ethanol, methanol,acetonitrile and acetone. In addition, there are also no particularlimitations on the pH of the extraction solution, and any arbitrary pHcan be used provided it does not deactivate the function of the targetexogenous protein.

There are no particular limitations on the method for isolating andpurifying the extracted exogenous protein, and ordinary proteinpurification methods can be used. For example, a target useful proteincan be purified and isolated by combining chromatography using a silicagel carrier, ion exchange carrier, gel permeation carrier, chelatingcarrier or pigment-loaded carrier and so forth, ultrafiltration, gelpermeation, dialysis, de-salting by salting out or concentration and soforth using an inherently possessed function as an indicator. Forexample, feline interferon-ω can be recovered in the soluble fractionobtained by homogenizing silk glands or cocoon and silk thread of asilkworm into which has been inserted a feline interferon-co gene with20 mM phosphate buffer (pH 7.0). Moreover, the purity of the felineinterferon-ω can be increased by adsorbing the resulting extract liquidonto, for example, a Blue Sepharose carrier and eluting the resultingbuffer solution containing the extract liquid after washing.

Cytokines produced in this manner can be used in pharmaceuticalapplications as well as various measurement and diagnostic applicationsin the same manner as cytokines produced with other conventionalproduction processes. In this case, they may also be used as a mixtureto which various additives have been added. In addition, the tissue orcocoon and silk thread of a silkworm in which cytokines have beenexpressed can also be used directly or after processing as fibers Formedical or clothing use. In addition, the tissue or silk thread of arecombinant silkworm in which enzymes have been expressed can be useddirectly in enzyme reactions.

EXAMPLES

Although the following provides a more detailed explanation of thepresent invention by indicating its examples, the present invention isnot limited to the descriptions of these examples.

Reference Example Method for Measuring Antiviral Activity

The physiological activity of interferon was measured according to thefollowing method as antiviral activity.

Antiviral activity was measured by the CPE method using vesicularstomatitis virus (VSV) for the virus, and using feline Fc9 cells (J. K.Yamamoto, et al.: vet. Immunol. and Immunopathol., 11, 1-19, 1986) forthe susceptive cells in the case of feline interferon-ω, or human FLcells in the case of human interferon-β. Namely, a sample diluent wasadded to the uppermost row of 96-well microtiter plate in whichsusceptive cells cultured at 37° C. to confluency, and then seriallydiluting in two-fold increments moving towards the lower end of theplate.

After culturing for 20 to 24 hours at 37° C., VSV was added followed byadditionally culturing for 16 to 20 hours at 37° C. Viable susceptivecells adhered to the microtiter plate were then stained withcrystal-violet stain containing 20% formalin, and as a result ofmeasuring the optical absorbance at 570 nm for the amount ofcrystal-violet remaining on the microplate, antiviral activity wasdetermined by comparison with a standard. Intercat (Toray) adjusted to1000 units/ml with cell culturing medium was used for the standard forfeline interferon-ω, while Feron (Toray) prepared to 1000 units/ml withcell culturing medium was used for the standard for human interferon-β.In addition, samples were used for the measurement of antiviral activityafter diluting 15-fold with cell culturing medium.

Example 1 Preparation of Bombyx mori Genomic DNA

Fifth instar third day silkworms were dissected to remove posterior silkgland tissue. After washing with 1×SSC, 200 μl of DNA extraction buffer(50 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0), 100 mM NaCl) were added.After adding Proteinase K (final concentration; 20 μg/ml) and adequatelygrinding up the tissue with a grinder, 350 μl of DNA extraction bufferand 60 μl of 10% SDS were added followed by incubating for 2 hours at50° C. After adding 500 μl of Tris-HCl-saturated phenol (pH 8.0) andmixing for 10 minutes, the supernatant was recovered by centrifuging for5 minutes at 4° C. and 10,000 rpm. After adding an equal volume ofphenol/chloroform/isoamyl alcohol (25:24:1) to the supernatant andmixing, the resulting mixture was centrifuged. Phenol/chloroform/isoamylalcohol was again added followed by centrifuging and recovery of thesupernatant. After adding an equal volume of chloroform/isoamyl alcohol(24:1) and mixing, the mixture was centrifuged. Chloroform/isoamylalcohol was again added to the resulting supernatant followed bycentrifuging and recovery of the supernatant. 1/10 volume 3 M sodiumacetate (pH 5.2) was then added to the resulting supernatant and mixedand, after additionally adding 2.5 volumes of cold ethanol and allowingit to stand undisturbed for 30 minutes at −80° C., the mixture wascentrifuged for 10 minutes at 4° C. and 15,000 rpm to precipitategenomic DNA. After washing the DNA precipitate with 70% ethanol, theprecipitate was air-dried. The precipitate was then dissolved in sterilewater containing RNase to 100 μg/ml to prepare a diluted genomic DNAsolution.

Example 2 Gene Preparation

The genes used were acquired by PCR by producing primers for thesequences on both ends using known sequences and using suitable DNAsources for the templates. Restriction sites were added to the ends ofthe primers for the subsequent gene construction procedure.

Feline interferon-ω gene (base numbers 9-593 of GenBank registration no.S62636) was acquired by PCR using two types of primers consisting ofprimer 3 (SEQ. ID No. 3) and primer 4 (SEQ. ID No. 4) and usingbaculovirus rBNV100 encoding feline interferon-w gene for the template.rBNV100 can be produced by, for example, cutting out FeIFN gene from aplasmid extracted from E. coli(pFeIFN1) (Patent Microorganism DepositoryNo. 1633), coupling to a silkworm cloning vector (T. Horiuchi, et al.,Agric. Biol. Chem., 51, 1573-1580, 1987), and co-transfecting silkwormestablished cells with the recombinant plasmid produced and silkwormnuclear polyhedrosis virus DNA.

Sericin-1 gene promoter (base numbers 599-1656 of GenBank registrationno. AB007831) was acquired by PCR using two types of primers consistingof primer 5 (SEQ. ID No. 5) and primer 6 (SEQ. ID No. 6) and usingsilkworm chromosomal DNA for the template. Fibroin H chain gene promoter(base numbers 255-574 of GenBank registration no. V00094) was acquiredby PCR using two types of primers consisting of primer 7 (SEQ. ID No. 7)and primer 8 (SEQ. ID No. 8) and using silkworm chromosomal DNA for thetemplate. Bovine growth hormone gene poly A (pcDNA3.1(+) sequencenumbers 1011-1253) was acquired by PCR using two types of primersconsisting of primer 9 (SEQ. ID No. 9) and primer 10 (SEQ. ID No. 10)and using plasmid pcDNA3.1(+) vector (Invitrogen) for the template.

PCR was carried out in accordance with the accompanying protocol usingKODplus (Toyobo). Namely, after adding 10 ng of each template in thecase of a plasmid or 100 ng in the case of chromosomal DNA, 30 pmol ofeach primer and 10 μl of the 10×PCR buffer provided, each reagent wasadded to a concentration of 1 mM MgCl₂, 0.2 mM dNTPs and 2 units ofKODplus followed by bringing up to a final volume of 100 μl. The PCRcomponents were then reacted for 30 cycles using a Perkin-Elmer DNAthermal cycler under DNA denaturation conditions of 94° C. for 15seconds, primer annealing conditions of 55° C. for 30 seconds, andelongation conditions of 68° C. for 30 to 60 seconds.

These reaction solutions were electrophoresed with 1 to 1.5% agarosegel, and DNA fragments consisting of a roughly 580 bp fragment in thecase of feline interferon-ω gene, a roughly 1 kbp fragment in the caseof sericin-1 promoter, a roughly 320 bp fragment in the case of fibroinH chain promoter, and a roughly 230 bp fragment in the case of bovinegrowth hormone poly A were extracted and prepared in accordance withordinary methods. After phosphorylating these DNA fragments withpolynucleotide kinase (Takara Shuzo), they were ligated to pUC19 vectorsubjected to dephosphorylation treatment after being cleaved with HincIIby reacting overnight at 16° C. using DNA Ligation Kit Ver. 2 (TakaraShuzo). These were then used to transform E. coli in accordance withordinary methods and the resulting transformants were confirmed tocontain the PCR fragments by performing PCR on the resulting coloniesunder the same conditions as previously described to prepare plasmids inwhich the PCR fragments were inserted according to ordinary methods.These plasmids were sequenced to confirm that the resulting fragmentsconsisted of the base sequences of each gene.

Example 3 Production of Plasmids for Gene Insertion

pigA3GFP (Nature Biotechnology 18, 81-84, 2000) was used for the plasmidfor gene insertion. Namely, vector pigA3GFP is a vector in which afterremoving a region encoding transposase from plasmid p3E1.2 disclosed inU.S. Pat. No. 6,2198,185, an A3 promoter (base numbers 1764-2595 ofGenBank registration no. U49854), GFP originating in pEGFP-N1 vector(Clontech) and poly A addition sequence originating in SV40 (basenumbers 659-2578 of GenBank registration no. U55762) are inserted intothat portion (Nature Biotechnology 18, 81-84, 2000). The expression unitof feline interferon-ω gene was inserted at the XhoI site upstream fromthe A3 promoter. The expression units of the inserted genes consisted ofa sericin-1 gene promoter-feline interferon-w-bovine growth hormone polyA addition sequence (SEQ. ID No. 1), or a fibroin H chain genepromoter-feline interferon-ω-bovine growth hormone poly A additionsequence (SEQ. ID No. 2). The following provides a detailed descriptionof the method.

Genes were cleaved from the plasmids prepared in Example 2 using therestrictase sites preset in the primers. Namely, insert fragments werecleaved using EcoRI and SalI in the case of sericin-1 gene promoter andfibroin H chain gene promoter, SalI and XbaI in the case of felineinterferon-ω, and XbaI and BamHI in the case of bovine growth hormonepoly A, followed by electrophoresing with 1 to 1.5% agarose gel andextracting and purifying the fragments in accordance with ordinarymethods.

200 ng of sericin-1 gene fragment, 100 ng of feline interferon-ω genefragment, and 50 ng of bovine growth hormone poly A were mixed andreacted overnight at 16° C. by adding an equal volume of DNA LigationKit Version 2 (Takara Shuzo). 0.5 μl of the reaction solution wassubjected to PCR using primer 11 (SEQ. ID No. 11) and primer 12 (SEQ. IDNo. 12) for 2 minutes of elongation under the same conditions as Example2. These reaction solutions were electrophoresed with 1% agarose gel,and the amplified, roughly 1.9 kb, DNA fragment (SIB fragment) wasextracted and purified in accordance with ordinary methods.

Similarly, 70 ng of fibroin H chain gene promoter fragment, 100 ng offeline interferon-ω gene fragment and 50 ng of bovine growth hormonepoly A were mixed and reacted overnight at 16° C. by adding an equalvolume of DNA Ligation Kit Version 2 (Takara Shuzo). 0.5 μl of thereaction solution was subjected to PCR using primer 13 (SEQ. ID No. 13)and primer 12 (SEQ. ID No. 12) for 2 minutes of elongation under thesame conditions as Example 1. These reaction solutions wereelectrophoresed with 1% agarose gel, and the amplified roughly 1.5 kbDNA fragment (FIB fragment) was extracted and purified in accordancewith ordinary methods.

After digesting these fragments with XhoI, they were ligated to pigA3GFPand subjected to XhoI treatment and dephosphorylation treatment byreacting overnight at 16° C. using DNA Ligation Kit Ver. 2 (TakaraShuzo). The plasmid containing the SIB fragment was designated as pigSIB(FIG. 1), and the plasmid containing the FIB fragment was designated aspigFIB (FIG. 2), and these were purified by centrifuging twice using thecesium chloride method and then used in a gene insertion experiment.

Example 4 Production of Gene Recombinant Silkworms (Fibroin H Chain GenePromoter)

The aforementioned pigFIB and helper plasmid pHA3PIG (FIG. 3, NatureBiotechnology 18, 81-84, 2000) were adjusted to a concentration of 200ng/ml each in 0.5 mM phosphate buffer (pH 7.0) and 5 mM KCl, after which15 to 20 nl were micro-injected into silkworm eggs within 4 hours afterbeing laid.

The larva that hatched from those silkworm eggs were raised, and theresulting adults (G0) were crossed within the same group. By observingthe resulting first generation (G1) individuals with fluorescence ofgreen fluorescence protein that had been simultaneously inserted withfeline interferon-co gene, those silkworms that contained the felineinterferon-ω gene in their chromosomes were screened. The ratios of themoth groups in which silkworms containing the inserted gene wereobtained are shown in Table 1. The silkworm eggs were injected twice,and gene recombinant silkworms were obtained from one moth group by thesecond injection. TABLE 1 Acquisition Status of Gene RecombinantSilkworms (Fibroin Heavy Chain Promoter) No. of moth groups No. ofpositive for No. of No. of sibling feline Experiment eggs eggs No. ofmated interferon-ω Group injected hatched adults moths gene 1 1215 292220 100 0 2 1326 374 250 123 1

The results of Southern blotting on the gene recombinant silkwormsobtained from that moth group are shown in FIG. 4. The method employedfor Southern blotting consisted of extracting chromosomal DNA from theG1 generation moths, electrophoresing restrictase-treated samples, anddetecting a membrane to which the DNA was transferred bychemiluminescence using the AlkPhos Direct Labeling and Detection System(Amersham-Pharmacia) using a nucleic acid probe specific for felineinterferon-ω.

When 11 G1 moths were investigated, feline interferon-ω gene wasconfirmed to have been inserted into 10 of the silkworms.

Example 5 Confirmation of Feline Interferon Production (Fibroin H ChainPromoter)

Since feline interferon-ω has antiviral activity, the presence of felineinterferon-ω can be determined according to its activity. Silkworms (G1)of the positive moth group obtained in Example 4 were mated with wildsilkworms, and the middle and posterior silk glands excised from fifthinstar larva of the resulting generation (G2) were confirmed to beinserted with feline interferon-w gene. These were then homogenizedusing 20 mM sodium phosphate buffer (pH 7.0), and the resulting extractwas measured using an antiviral activity measuring system that usedfeline cells. As a result, although antiviral activity was detected forboth middle silk glands and posterior silk glands from the silk glandextracts of gene-containing silkworms, activity was not detected fromthe silk gland extracts of wild silkworms used as the control. Thoseresults are shown in FIG. 5.

Feline interferon-ω is thought to mainly be expressed in posterior silkglands under the control of fibroin H chain promoter. It is believed tosubsequently migrate into the middle and anterior silk glands in thesame manner as fibroin, and the distribution of physiological activityis considered to coincide with this. On the other hand, there was noantiviral activity detected from silkworms into which the gene was notinserted. This clearly demonstrates that feline interferon-ω protein isexpressed while retaining its physiological activity in silkworms intowhich feline interferon-ω gene has been inserted.

Example 6 Purification of Feline Interferon

Feline interferon was purified from the extract of posterior silk glandsexcised from G2 generation, fifth instar silkworms obtained in Example5. 1 ml of extract was passed through a HiTrap Blue Sepharose column(Amersham-Pharmacia) followed by washing the column with 10 ml of 20 mMsodium phosphate buffer (pH 7.0). Continuing, the column was eluted with10 ml of 20 mM sodium phosphate buffer (pH 8.0)-0.5 M NaCl and then with10 ml of 20 m sodium phosphate buffer (pH 8.0)-1 M NaCl.

The washing fraction, 0.5 M elution fraction and 1 M elution fractionwere collected, desalted and concentrated to about 1 ml. The results ofdetermining the antiviral activity and amount of protein of the extractand each purified fraction are shown in Table 2. TABLE 2 Purification ofFeline Interferon-ω by Blue Sepharose Chromatography Antiviral Amt. ofSpecific activity protein activity (U/ml) (mg/ml) (U/mg) Extracted 5230.37 1401 sample Blank, 23 2.91 8 washing fraction 0.5 M NaCl 1494 0.851758 elution fraction   1 M NaCl >6270 0.41 >15293 elution fraction

As a result of the purification procedure, antiviral activity, namelyfeline interferon-α, could be recovered in the 1 M elution fraction, andits specific activity was roughly 10 times that of the extract.

Example 7 Production of Recombinant Gene Silkworms (Sericin-1 Promoter)

The aforementioned pigSIB and a helper plasmid were adjusted to aconcentration of 200 ng/ml each in 0.5 mM phosphate buffer (pH 7.0) and5 mM KCl, after which 15 to 20 nl were micro-injected into silkworm eggswithin 4 hours after being laid. The larva that hatched from thosesilkworm eggs were raised, and the resulting adults (G0) were crossedwithin the same group. Insertion of feline interferon-ω gene into thechromosomes was investigated by observing the fluorescence of greenfluorescence protein from the resulting first-generation (G1)individuals. In two experiments, a gene recombinant vector containingfeline interferon-ω gene coupled to sericin promoter was micro-injectedinto 1218 and 1375 eggs, respectively, and 12 positive moth groups eachwere able to be obtained (Table 3). TABLE 3 Acquisition Status of GeneRecombinant Silkworms (Sericin Promoter) No. of moth groups No. ofpositive for No. of No. of sibling feline Experiment eggs eggs No. ofmated interferon-ω Group injected hatched adults moths gene 1 1218 500320 158 12 2 1375 540 500 225 12

One silkworm (G1) each that was confirmed to contain the gene wasselected from 3 moths groups in the first experiment and 2 moth groupsin the second experiment among the resulting positive moth groups, andgenomic DNA was extracted from their silk glands. After treating withEcoRI or BglII, Southern blotting analysis was performed on the DNAusing feline interferon-o gene as a probe. Those results are shown inFIG. 6. As a result, feline interferon-ω gene was confirmed to beinserted in the genomes of all silkworms. In addition, the site at whichthe gene was inserted into the genome was determined to be differentdepending on the moth group due to differences in the detected signalsize.

Next, mRNA expression of feline interferon-ω gene was investigated.Seven G1 silkworms confirmed to contain feline interferon-co gene bySouthern blotting were randomly selected, their mRNA was extracted andthe expression of feline interferon-o gene mRNA was investigated byRT-PCR. Isogen (Nippon Gene) and Oligodex dT-30 (Roche Diagnostics) wereused for mRNA extraction and purification, the Ready-To-Go T-PrimedFirst-Strand Kit (Amersham-Pharmacia) was used for cDNA synthesis, andthe procedure was carried out according to the protocol provided withthe kit. As a result of carrying out PCR under the same conditions asduring the acquisition of feline interferon-ω gene of Example 2,expression of feline interferon-ω gene mRNA was confirmed for all of thesilkworms (FIG. 7).

Example 8 Confirmation of Feline Interferon Production in Middle SilkGlands and Cocoon and Silk Thread

The middle silk glands were excised from three gene recombinantsilkworms obtained in Example 7 and one wild silkworm followed byhomogenizing using 20 mM sodium phosphate buffer (pH 7.0) andcentrifuging to prepare extracts. In addition, one cocoon each from thegene recombinant silkworms and wild silkworm were extracted in the samemanner. When these extracts were measured for their antiviral activity,antiviral activity was detected in the middle silk glands of all of thegene recombinant silkworms, but was not detected in the silk glands ofthe wild silkworm. Moreover, antiviral activity was also detected in thecocoons of the gene recombinant silkworms (FIG. 8).

On the basis of these findings, feline interferon-co was determined tobe expressed in gene recombinant silkworms while retaining itsphysiological activity, and that activity was determined to remain inthe silk thread.

Example 9 Production of Plasmids for Insertion of Human Interferon-βGene

Production of plasmids for insertion of human interferon-β gene wascarried out according to the same method as the case of felineinterferon-ω gene indicated in Examples 2 through 4.

Namely, PCR was carried out using primer 14 (SEQ. ID No. 14) and primer15 (SEQ. ID No. 15) and using plasmid pORF-hIFN-β encoding humaninterferon-β gene as a template to obtain a human interferon-β genefragment. After treating this fragment with restriction endonucleasesSalI and XbaI, a plasmid was constructed that contained a geneexpression sequence in which fibroin H chain gene promoter was coupledto the 5′ terminal or poly A signal originating in bovine growth hormonegene was coupled to the 3′ terminal (fibroin H chain promoter-humaninterferon-β gene-bovine growth hormone gene poly A signal (FhIB): SEQ.ID No. 16, sericin gene promoter-human interferon-β-bovine growthhormone gene poly A signal (ShIB); SEQ. ID No. 17).

The aforementioned FhIB and ShIB sequences for gene expression were thenrespectively cleaved from these plasmids by treating with XhoI, andcoupled to pigA3GFP that had been subjected to dephosphorylationtreatment after being cleaved with XhoI. The plasmid containing the FhIBfragment was designated as pigFhIB, while the plasmid that contained theShIB fragment was designated as pigShIB. These fragments were purifiedby centrifuging twice according to the cesium chloride method and thenused in a gene insertion experiment.

Example 10 Production of Human Interferon-β Gene Recombinant Silkworms

Production of human interferon-β gene recombinant silkworms was carriedout according to the same method used to produce feline interferon-ωgene recombinant silkworms indicated in Example 4 by using the geneinsertion plasmids produced in Example 9.

Namely, pigFhIB and pigShIB were respectively micro-injected intosilkworm eggs together with helper plasmid pHA3PIG, and the resultingadults were crossed followed by screening of the next generation. Wheneach plasmid was injected into 600 eggs, silkworms positive for greenfluorescence were obtained in 7 moth groups for silkworms containingpigFhIB and in 5 moth groups for silkworms containing pigShIB, andinsertion of the genes into their chromosomes was confirmed by PCR. Thesilk glands and silk thread were harvested from these silkworms andtheir extracts were used to measure the physiological activity of humaninterferon-β in the form of their antiviral activity. The values areshown in the table after having been corrected for total proteinconcentrations in the samples. TABLE 4 Antiviral Activity in TissueExtracts of Human Interferon-β Gene Recombinant Silkworms Moth groupAntiviral activity (units/g no. - protein) Individual Posterior Middlesilk Promoter no. silk glands glands Silk thread Fibroin H 3-1 59972382591 Not tested chain 3-2 656110 41545 11-1  502750 19859 11-2  11556039130 Sericin 1-1 Not tested 53884 42187 1-2 648953 101713 5-1 437291133288 5-2 541106 92749 Normal silkworms Not Not Not detected detecteddetected

Detection limit. Approx. 1000 units/g protein.

As a result, since antiviral activity was detected from the posteriorand middle silk glands of the silkworms inserted with pigFhIB andantiviral activity was detected from the middle silk glands and silkthread of the silkworms inserted with pigShIB, interferon-β wasconfirmed to be produced in the silk gland tissue of these silkworms.

Example 11 Production of Plasmids for Insertion of Feline GranulocyteColony Stimulating Factor Gene

Production of plasmids for insertion of feline granulocyte colonystimulating factor gene was carried out according to the same method asthe case of feline interferon-co gene indicated in Examples 2 through 4.

Feline granulocyte colony stimulating factor gene was obtained in theform of a feline granulocyte colony stimulating factor gene fragment bycarrying out PCR using primer 18 (SEQ. ID No. 18) and primer 19 (SEQ. IDNo. 19) from cDNA obtained from CRFX cells stimulated for 24 hours withLPS at 10 μg/ml according to the report of Yamamoto, et al. (Gene, 274,263-269, 2001). After treating this fragment with SalI and XbaI,plasmids were constructed that contained a sequence for gene expressionin which fibroin H chain gene promoter or sericin gene promoter wascoupled to the 5′ terminal or poly A signal originating in bovine growthhormone gene was coupled to the 3′ terminal (fibroin H chainpromoter-feline granulocyte colony stimulating factor gene-bovine growthhormone gene poly A signal (FGB): SEQ. ID No. 20, sericin genepromoter-feline granulocyte colony stimulating factor gene-bovine growthhormone gene poly A signal (SGB): SEQ. ID No. 21).

The aforementioned FGB and SGB sequences for gene expression were thenrespectively cleaved from these plasmids by treating with XhoI, andcoupled to pigA3GFP that had been subjected to dephosphorylationtreatment after being cleaved with XhoI. The plasmid containing the FGBfragment was designated as pigFGB, while the plasmid that contained theSGB fragment was designated as pigSGB. These fragments were purified bycentrifuging twice according to the cesium chloride method and then usedin a gene insertion experiment.

Example 12 Production of Feline Granulocyte Colony Stimulating FactorGene Recombinant Silkworms

Production of feline granulocyte colony stimulating factor generecombinant silkworms was carried out according to the same method usedto produce feline interferon-ω gene recombinant silkworms indicated inExample 4 by using the gene insertion plasmids produced in Example 11.

Namely, pigFhIB and pigShIB were respectively micro-injected intosilkworm eggs together with helper plasmid pHA3PIG, and the resultingadults were crossed followed by screening of the next generation. Wheneach plasmid was injected into 600 eggs, silkworms positive for greenfluorescence were obtained in 3 moth groups for silkworms containingpigFGB and in 7 moth groups for silkworms containing pigSGB, andinsertion of the genes into their chromosomes was confirmed by PCR. Thesilk glands and silk thread were harvested from these silkworms andtheir extracts were used to measure the physiological activity of felinegranulocyte colony stimulating factor in the form of growth promotingactivity of NFS-60 cells (ATCC).

Measurement of growth promoting activity was carried out in the mannerdescribed below. First, NFS-60 cells were seeded in a 96-well plate inthe absence of M-CSF at 2×104 cells/well followed 30 minutes later bythe addition of 10 μl of sample. After culturing for an additional 24hours, cell growth activity was measured using the Cell Counting Kit-8(Dojindo). The amount of sample that yielded 50% of the maximum growthpromoting effect (ED50) was defined as 1 unit/ml, and the physiologicalactivity in the sample was calculated by multiplying by the dilutionfactor. The values are shown in the table after having been correctedfor total protein concentrations in the samples. TABLE 5 GrowthPromoting Activity in Tissue Extracts of Feline Granulocyte ColonyStimulating Factor Gene Recombinant Silkworms Moth group Growthpromoting activity (units/g no. - protein) Individual Posterior Middlesilk Promoter no. silk glands glands Silk thread Fibroin H 9-1 36 NotNot tested chain 9-2 412 detected 16-1  326 113 16-2  4039 226 53Sericin 3-1 Not tested 4330 590 3-2 2277 524 8-1 3966 846 8-2 2137 211Normal Silkworms Not Not Not detected detected detected

Detection limit; Approx. 20 units/g protein.

As a result, as growth promoting activity was detected from theposterior and middle silk glands of the silkworms inserted with pigFGBand growth promoting activity was detected from the middle silk glandsand silk thread of the silkworms inserted with pigSGB, felinegranulocyte colony stimulating factor was confirmed to be produced inthe silk gland tissue of these silkworms.

Example 13 Gene Preparation

A study was conducted on improving production amounts in silk glandtissue and silk thread using feline interferon-ω as a model of aphysiologically active protein.

The genes used were acquired by PCR by producing primers for thesequences on both ends using known sequences and using suitable DNAsources for the templates. Restrictase sites were added to the ends ofthe primers for subsequent gene manipulation.

Fibroin H chain promoter (base numbers 62118-62437 of GenBankregistration no. AF226688: to be referred to as the P region) wasacquired by PCR using two types of primers consisting of primer 25 (SEQ.ID No. 25) and primer 26 (SEQ. ID No. 26) and using Bombyx mori genomicDNA for the template.

Fibroin H chain promoter-fibroin H chain gene first exon-firstintron-second exon region (base numbers 62118-63513 of GenBankregistration no. AF226688: to be referred to as the HP region) wasacquired by using two types of primers consisting of primer 25 (SEQ. IDNo. 25) and primer 31 (SEQ. ID No. 31) and using Bombyx mori genomic DNAfor the template.

Fibroin H chain upstream promoter-fibroin H chain gene first exon-firstintron region (base numbers 57444-62927 of GenBank registration no.AF226688: to be referred to as the HUP region) was acquired by PCR usingtwo types of primers consisting of primer 33 (SEQ. ID No. 33) and primer34 (SEQ. ID No. 34) and using Bombyx mori genomic DNA for the template.

Feline interferon-ω gene (base numbers 9-593 of GenBank registration no.S62636: to be referred to as the IC region) was acquired by PCR usingtwo types of primers consisting of primer 27 (SEQ. ID No. 27) and primer28 (SEQ. ID No. 28) and using baculovirus rBNV100, which encodes felineinterferon-ω gene, for the template. rBNV100 can be produced by, forexample, cutting out feline interferon-w gene from a plasmid extractedfrom E. coli (pFeIFN1) (Patent Microorganism Depository No. 1633),coupling to a silkworm cloning vector (T. Horiuchi, et al., Agric. Biol.Chem., 51, 1573-1580, 1987), and co-transfecting silkworm establishedcells with the recombinant plasmid produced and silkworm nuclearpolyhedrosis virus DNA.

Fibroin H chain poly A signal region (base numbers 79201-79995 ofGenBank registration no. AF226688: to be referred to as the A region)was acquired by PCR using two types of primers consisting of primer 29(SEQ. D No. 29) and primer 30 (SEQ. ID No. 30) and using Bombyx morigenomic DNA for the template.

Fibroin H chain C terminal region gene-fibroin H chain poly A signalregion (base numbers 79099-79995 of GenBank registration no. AF226688:to be referred to as the HA region) was acquired by PCR using two typesof primers consisting of primer 32 (SEQ. ID No. 32) and primer 30 (SEQ.ID No. 30) and using Bombyx mori genomic DNA for the template.

β-galactosidase (β-gal) gene was acquired by PCR using two types ofprimers consisting of primer 37 (SEQ. ID No. 37) and primer 38 (SEQ. IDNo. 38) and using pβgal-Basic vector (Clontech) for the template.

PCR was carried out in accordance with the accompanying protocol usingKODplus (Toyobo). Namely, after adding 100 ng of each template in thecase of Bombyx mori genomic DNA or 10 ng in the case of Bombyx moriposterior silk gland cDNA and pβgal-Basic vector, 50 pmol of each primerand 10 μl of the 10×PCR buffer provided, each reagent was added to aconcentration of 1 mM MgCl₂, 0.2 mM dNTPs and 2 units of KODplusfollowed by bringing to a final volume of 100 μl. The PCR componentswere then reacted for 30 cycles using a Perkin-Elmer DNA thermal cyclerunder DNA denaturation conditions of 94° C. for 15 seconds, primerannealing conditions of 55° C. for 30 seconds, and elongation conditionsof 68° C. for 60 to 300 seconds.

These reaction solutions were electrophoresed with 1 to 1.5% agarosegel, and a DNA fragment of roughly 0.3 kbp in the P region, roughly 1.4kbp in the HP region, roughly 5.5 kbp in the HUP region, roughly 580 bpin the IC region, roughly 0.8 bp in the A region, roughly 0.9 bp in theHA region and roughly 3.2 kbp in the β-gal gene were extracted andprepared in accordance with ordinary methods. After phosphorylatingthese DNA fragments with polynucleotide kinase (Takara Shuzo), they wereligated to pUC19 vector subjected to dephosphorylation treatment afterbeing cleaved with HincII by reacting overnight at 16° C. using DNALigation Kit ver. 2 (Takara Shuzo). These were then used to transform E.coli in accordance with ordinary methods and the resulting transformantswere confirmed to be inserted with PCR fragments by performing PCR onthe resulting colonies under the same conditions as previously describedto prepare plasmids in which the PCR fragments were inserted accordingto ordinary methods. These plasmids were sequenced to confirm that theresulting fragments consisted of the base sequences of each gene.

Example 14 Production of Plasmids for Expression of β-Galactosidase

The plasmid retaining β-gal gene prepared in Example 13 was cleaved withSalI and HindIII followed by insertion therein of a roughly 0.3 kbpfragment (P region) cleaved by SalI and HindIII from a plasmid retainingfibroin H chain promoter. Moreover, this was then cleaved with BamHIfollowed by insertion therein of a roughly 0.8 kbp region (A region)cleaved with BamHI from a plasmid having a fibroin H chain poly A signalregion, and purifying the resulting plasmid retaining the β-gal geneusing the Qiagen Plasmid Maxi Kit in accordance with the protocolprovided. The resulting plasmid was named pPga1A, and it was confirmedto be the target plasmid by PCR and sequencing.

Similarly, the plasmid retaining the β-gal gene prepared in Example 13was cleaved with SalI and HindIII followed by insertion therein of aroughly 1.4 kbp fragment (HP region) cleaved with SalI and HindIII froma plasmid retaining the fibroin H chain promoter-fibroin H chain genefirst exon-first intron-second exon region. Moreover, this was thencleaved with BamHI followed by insertion therein of a roughly 0.9 kbpfragment (HA region) cleaved with BamHI from a plasmid retaining afibroin H chain C terminal region-fibroin H chain poly A signal region,and purifying the resulting plasmid retaining the β-gal gene using theQiagen Plasmid Maxi Kit in accordance with the protocol provided. Theresulting plasmid was named pHPgalHA, and it was confirmed to be thetarget plasmid by PCR and sequencing.

Example 15 Production of Plasmids for Gene Insertion

pigA3GFP (Nature Biotechnology 18, 81-84, 2000) was used for the plasmidfor gene insertion. Namely, vector pigA3GFP is a vector in which afterremoving a region encoding transposase from plasmid p3E1.2 disclosed inU.S. Pat. No. 6,218,185, an A3 promoter (base numbers 1764-2595 ofGenBank registration no, U49854), GFP originating in pEGFP-N1 vector(Clontech) and poly A addition sequence originating in SV40 (basenumbers 659-2578 of GenBank registration no. U55762) are inserted intothat portion. The XhoI site located upstream from the A3 promoter wasblunt ended followed by insertion of an expression cassette of felineinterferon-ω gene. The constitution of the gene expression cassette usedin the present example consisted of fibroin H chain promoter-felineinterferon-O-fibroin H chain C terminal region-fibroin H chain poly Asignal region (HP-IC-HA), or fibroin H chain upstream promoter-fibroin Hchain gene first exon-first intron-second exon region-felineinterferon-ω-fibroin H chain C terminal region-fibroin H chain poly Asignal region (HUP-IC-HA), or fibroin H chain promoter-fibroin H chaingene first exon-first intron-second exon region-felineinterferon-ω-fibroin H chain poly A signal region (HP-IC-A).

The following indicates the specific method employed.

The P-IC-A construct was produced according to the following procedure.The plasmid retaining feline interferon-ω (IC region) prepared inExample 13 was cleaved with SalI and HindIII followed by insertiontherein of a roughly 0.3 kbp fragment (P region) cleaved with SalI andHindIII from a plasmid retaining fibroin H chain promoter. Moreover,this was cleaved with BamHI followed by insertion therein of a roughly0.8 kbp fragment (region A) cleaved with BamHI from a plasmid retainingfibroin H chain poly A signal region. This plasmid retaining P, IC and Awas cleaved with AscI and the cleaved roughly 1.7 kbp fragment was bluntended with T4 DNA Polymerase (Takara Shuzo) and coupled to blunt endedand dephosphorylated pigA3GFP XhoI site to produce a construct for geneinsertion containing the P-IC-A gene cassette. The procedure is shown inFIGS. 9 and 10.

The HP-IC-HA construct was produced in the following manner. The plasmidretaining feline interferon-ω (IC region) prepared in Example 13 wascleaved with SalI and HindIII followed by insertion therein of a roughly1.4 kbp fragment (HP region) cleaved with SalI and HindIII from aplasmid retaining a fibroin H chain promoter-fibroin H chain gene firstexon-first intron-second exon region. Moreover, this was cleaved withBamHI followed by insertion therein of a roughly 0.9 kbp fragment(region HA) cleaved with BamHI from a plasmid retaining fibroin H chainC terminal region-fibroin H chain poly A signal region. This plasmidretaining HP, IC and HA was cleaved with AscI and the cleaved roughly2.9 kbp fragment was blunt ended with T4 DNA Polymerase (Takara Shuzo)and coupled to blunt ended and dephosphorylated pigA3GFP XhoI site toproduce a construct for gene insertion containing the HP-IC-HA genecassette. The procedure is shown in FIGS. 11 and 12.

The HUP-IC-HA construct was produced according to the followingprocedure. A roughly 2.1 kbp fibroin H chain first intron-second exonregion-feline interferon-ω-fibroin H chain C terminal region-fibroin Bchain poly A signal region was acquired by PCR using two types ofprimers consisting of primer 35 (SEQ. ID No. 35) and primer 36 (SEQ. IDNo. 36) and using 1 ng of HP-TC-HA construct for the template. This wasthen cleaved with XhoI and SphI followed by insertion therein of aroughly 5.5 kbp fragment (HUP region) cleaved with XhoI and SphI from aplasmid retaining fibroin H chain upstream promoter-fibroin H chain genefirst exon-first intron. This plasmid retaining HUP, IC and HA wascleaved with AscI and the cleaved roughly 7.6 kbp fragment was bluntended with T4 DNA Polymerase (Takara Shuzo) and coupled to blunt endedand dephosphorylated pigA3GFP XhoI site to produce a construct for geneinsertion containing the HUP-IC-HA gene cassette. The procedure is shownin FIGS. 13 and 14.

The HP-IC-A construct was produced according to the procedure describedbelow. The plasmid retaining feline interferon-ω (IC region) prepared inExample 13 was cleaved with SalI and HindIII followed by insertiontherein of a roughly 1.4 kbp fragment (HP region) cleaved with SalI andHindIII from a plasmid retaining fibroin H chain promoter-fibroin Hchain first exon-first intron-second exon region. Moreover, this wascleaved with BamHI followed by insertion therein of a roughly 0.8 kbpfragment (region A) cleaved with BamHI from a plasmid retaining fibroinH chain poly A signal region. This plasmid retaining HP, IC and A wascleaved with AscI and the cleaved roughly 2.8 kbp fragment was bluntended with T4 DNA Polymerase (Takara Shuzo) and coupled to blunt endedand dephosphorylated pigA3GFP XhoI site to produce a construct for geneinsertion containing the HP-IC-A gene cassette. The procedure is shownin FIGS. 15 and 16.

The P-IC-A gene insertion construct, HP-IC-HA gene insertion construct,HUP-IC-HA gene insertion construct and HP-IC-A gene insertion constructwere purified using the Qiagen Plasmid Maxi Kit in accordance with theprotocol provided.

Example 16 Expression of β-Galactosidase in Silkworm Silk Gland

Gold Particles having a diameter of 1.6 μm were washed and sterilizedwith 100% ethanol and then suspended in sterilized distilled water (60mg/ml). Incorporation of β-gal gene expression cassettes into silkwormsilk glands was carried out using a gene gun. Namely, 50 μl (0.3 mg) ofgold particles, 10 μg of expression plasmid pPgalA or pHPgalHA obtainedin Example 14, 50 μl of 2.5 M calcium chloride and 20 μl of 0.1 Mspermidine were successively mixed and after allowing to stand for 30minutes at room temperature, the mixture was centrifuged to recover thegold particles coated with pHgalC. After washing the resulting metalparticles twice with 70% ethanol, they were dispersed in 50 μl of 100%ethanol. 10 μl of the suspension of gold particles were placed on amicrocarrier and dried. The Model PDS-1000/He (Bio-Rad) was used for thegene gun. The posterior silk glands excised from fifth instar third daysilkworm larva were gently washed twice with PBS, placed on a 1% agarplate and sprayed with gold particles coated with DNA at a pressure of1,100 psi. Following insertion of DNA, the silk glands were transferredto 20 ml of Grace's insect medium and cultured for 2 days at 25° C.After culturing, the culture supernatant and silk gland cells wererecovered and confirmed for the expression of β-gal.

Expression was confirmed by Western analysis, The silk gland cells werehomogenized in PBS to extract the cell contents. The culture supernatantand cell extract were both adjusted to a total protein concentration of1.0 mg/ml, and these were then used as samples for SDS-PAGE. Afterblotting onto a membrane, β-gal protein was detected using the ECL Plus™Western Blotting Kit (Amersham-Pharmacia) in accordance with theprotocol provided. Namely, the blotted membrane was first blockedovernight at 4° C. in blocking solution (5% skim milk, 0.1%Tween20/PBS). The membrane was then washed twice with TPBS (0.1% Tween20/PBS) and treated for 1 hour at room temperature with anti-β-galprotein antibody (Sigma) diluted 1000-fold with TPBS. The membrane waswashed twice with TPBS and additionally washed three times with TPBS for5 minutes each. After diluting 10000-fold with TPBS, the membrane wastreated for 1 hour at room temperature with HRP-labeled anti-rabbit IgGantibody. After washing the membrane twice with TPBS and then threetimes with TPBS for 5 minutes each, the detection reagents of the ECLPlus™ Western Blotting Detection System (Amersham-Pharmacia) were added(Solution A+Solution B). Luminescence was then exposed ontoHyperfilmTMECL™ and developed.

Since β-gal protein was only detected in the silk gland cells andculture supernatant containing pHPgalHA, it was clearly determined thata region other than fibroin H chain promoter, namely fibroin H chaingene first exon-first intron-second exon region, plays an important rolein protein synthesis or gene expression within the cells. In addition,secretion outside the cells was also confirmed. Those results are shownin FIG. 17.

Example 17 Production of Recombinant Gene Silkworms

Each of the gene insertion plasmids described in Example 15 and DNA thatproduces piggyBac transposase protein (pHA3PIG) were adjusted to aconcentration of 200 μg/ml each in 0.5 mM phosphate buffer (pH 7.0) and5 mM KCl, after which 3 to 20 nl were micro-injected into silkworm eggswithin 4 hours after being laid.

The larva that hatched from those silkworm eggs were raised, and theresulting adults (G0) were crossed within the same group. By observingthe resulting first generation (G1) individuals for fluorescence ofjellyfish green fluorescence protein, those silkworms that contained thejellyfish green fluorescence protein gene in their chromosomes werescreened. As a result, gene recombinant silkworms were obtained thatemitted fluorescent light due to the action of the jellyfish greenfluorescence protein.

Example 18 Expression Analysis of Recombinant Protein in Silk GlandTissue by Western Analysis

The expression of feline interferon-ω in tissue was investigated byWestern analysis after recovering the posterior silk gland tissue fromnon-transformed silkworms and transformed silkworms (HP-IC-A transformedsilkworms, HP-IC-EA transformed silkworms and HUP-IC-HA transformedsilkworms). The silkworm posterior silk glands were homogenized in 100mM sodium phosphate buffer (pH 7.0), and the supernatant was recoveredfollowing centrifugation for use as samples. Feline interferon was thendetected using the ECL Plus™ Western Blotting Kit (Amersham-Pharmacia)in accordance with the protocol provided. Namely, the blotted membranewas blocked overnight at 4° C. in blocking solution (5% skim milk, 0.1%Tween20/PBS). The membrane was then washed twice with TPBS (0.1% Tween20/PBS) and treated for 1 hour at room temperature with anti-felineinterferon antibody diluted 1000-fold with TPBS. The membrane was washedtwice with TBPS and additionally washed three times with TPBS for 5minutes each. After diluting 10000-fold with TPBS, the membrane wastreated for 1 hour at room temperature with HRP-labeled anti-rabbit IgGantibody. After washing the membrane twice with TPBS and then threetimes with TPBS for 5 minutes each, the detection reagents of the ECLPlus™ Western Blotting Detection System (Amersham-Pharmacia) were added(Solution A+Solution B). Luminescence was then exposed ontoHyperfilmTMECL™ and developed. As a result, in contrast to signals notbeing detected from posterior silk gland tissue of the non-transformedsilkworms and P-IC-A construct transformed silkworms, signals weredetected from the posterior silk gland tissue of transformed silkwormscontaining the EP-IC-A construct, HP-IC-HA construct and HUP-IC-HAconstruct. Based on the results of this experiment, a region other thanthe fibroin H chain promoter, namely the fibroin H chain gene firstexon-first intron-second exon region, was reconfirmed to play animportant role in drastically improving protein synthesis or geneexpression within silkworm posterior silk gland cells. These results areshown in FIG. 18. The accumulated amount of feline interferon inposterior silk gland tissue in the transformed silkworms containing theHUP-IC-HA gene cassette that contains the 5.5 kbp fibroin H chain 5′terminal region, feline interferon gene and fibroin H chain 3′ terminalwas higher than the accumulated amount of feline interferon in posteriorsilk gland tissue in transformed silkworms containing the HP-IC-HA genecassette that contains the fibroin H chain 5′ terminal promoter region,feline interferon gene and fibroin H chain 3′ terminal. A gene regionthat improved the amount of protein produced is thought to be present ina region upstream from the E chain 5′ terminal.

Example 19 Measurement of Recombinant Protein in Silk Thread by WesternAnalysis

Next, the secretion of exogenous protein, namely feline interferon-ω, insilk thread was investigated.

10 mg each of the cocoons from non-transformed silkworms and transformedsilkworms (transformed silkworms containing the HP-IC-A gene,transformed silkworms containing the HP-IC-HA gene, and transformedsilkworms containing the HUP-IC-HA gene) were weighed out, and afteradding 4 ml of 60% LiSCN and stirring, the cocoons were dissolved byallowing to mix overnight at room temperature. The dissolved cocoonswere then diluted 10-fold with 8 M urea, 2% SDS and 5% 2-mercaptoethanolto prepare samples. The levels of feline interferon in the samples werethen detected using the ECL Plus™ Western Blotting Kit(Amersham-Pharmacia) in accordance with the protocol provided. Thoseresults were then measured for signal intensity using a Molecular Imager(Bio-Rad) and the protein contents were measured by comparing with thesignal intensities of known concentrations of feline interferon.

As a result, in contrast to signals not being detected from the cocoonsof the non-transformed silkworms and transformed silkworms containingHP-IC-A gene, signals were detected from the cocoons of transformedsilkworms containing HP-IC-HA gene and transformed silkworms containingHUP-IC-HA gene, thereby confirming that feline interferon protein issecreted into silk thread. In addition, the content was about 0.8 to2.0% in HP-IC-HA transformed silkworms, and about 1.8 to 5.4% inHUP-IC-HA transformed silkworms. This is equivalent to 0.4 to 2 mg interms of the weight per silkworm.

On the basis of the results of this experiment, the 3′ terminal portionof fibroin H chain gene was clearly demonstrated to play an importantrole in the secretion of protein synthesized in posterior silk glandcells into silk thread. These results are shown in FIG. 19. The amountof feline interferon produced in transformed silkworms containing theHUP-IC-HA gene cassette that contains a 5.5 kbp fibroin H chain 5′terminal promoter region, feline interferon gene and fibroin H chain 3′terminal was higher than the amount of feline interferon produced intransformed silkworms containing the HP-IC-HA gene cassette thatcontains fibroin H chain 5′ terminal promoter region, feline interferongene and fibroin H chain 3′ terminal. A gene region that improves theamount of protein produced is considered to be present in a regionupstream from the H chain 5′ terminal.

Example 20 Measurement of Recombinant Protein in Silk Thread by ELISA

Quantitative determination of feline interferon-o in silk thread wascarried out.

10 μg each of the cocoons from non-transformed silkworms and transformedsilkworms (transformed silkworms containing the HP-IC-A gene,transformed silkworms containing the HP-IC-HA gene, and transformedsilkworms containing the HUP-IC-HA gene) were weighed out, and afteradding 4 ml of 60% LiSCN and stirring, the cocoons were dissolved byallowing them to mix overnight at room temperature. The dissolvedcocoons were diluted 8-fold or 16-fold with PBS and applied to amicrotiter plate. Known concentrations of feline interferon seriallydiluted with PBS were used for the standards.

As a result, feline interferon-w was not detected in silk thread intransformed silkworms containing HP-IC-A gene, but was detected at about1.1 to 2.2% in transformed silkworms containing HP-IC-HA gene, and atabout 1.0 to 4.9% in transformed silkworms containing HUP-IC-HA gene.

INDUSTRIAL APPLICABILITY

A cytokine could be recovered in large volume while retaining itsphysiological activity from the silk glands or silk thread of generecombinant silkworms obtained by producing a plasmid vector, in which acytokine gene is coupled to a promoter that functions in silkworm silkglands, and then inserting those genes into silkworm chromosomes. Inaddition, the resulting cytokine extract has a low level ofcontaminating proteins, and can be purified easily as compared withmethods of the prior art.

A large amount of exogenous protein could be produced within silk glandcells, outside silk gland cells and in silk thread by inserting anexpression gene cassette, in which the DNA sequence of the 5′ terminalportion and the DNA sequence of the 3′ terminal portion of fibroin Hchain gene were fused to an exogenous protein gene, into silk glandcells and so forth. The use of this novel technique led to theestablishment of a technology for producing easily purified exogenousprotein by producing exogenous protein using silk glands without the useof a recombinant baculovirus.

1. A process for producing recombinant cytokine comprising producing agene recombinant silkworm that incorporates cytokine gene in itschromosomes, producing recombinant cytokine protein in the silk glandsor cocoon and silk thread of the resulting gene recombinant silkworm,and recovering the cytokine from the silk glands or cocoon and silkthread.
 2. A process for producing recombinant cytokine according toclaim 1 wherein a cytokine gene coupled downstream from a promoterspecifically expressed in silk glands is incorporated in a chromosome.3. A process for producing recombinant cytokine according to claim 2wherein the promoter specifically expressed in silk glands is a sericingene promoter.
 4. A process for producing recombinant cytokine accordingto claim 2 wherein the promoter specifically expressed in silk glands isa fibroin H chain gene promoter.
 5. A process for producing recombinantcytokine according to any of claims 1 through 4 wherein cytokine gene isincorporated in silkworm chromosomes using DNA originating in atransposon.
 6. A process for producing recombinant cytokine according toclaim 5 wherein the cytokine gene is located between a pair of invertedterminal sequences originating in a transposon.
 7. A process forproducing recombinant cytokine according to claim 5 or 6 wherein the DNAoriginating in a transposon originates in an insect.
 8. A process forproducing recombinant cytokine according to claim 7 wherein thetransposon originates in piggyBac transposon originating in alepidopteron.
 9. A process for producing recombinant cytokine accordingto any of claims 1 through 8 wherein the cytokine gene is interferongene or colony stimulating factor gene.
 10. A process for producingrecombinant cytokine according to claim 9 wherein the interferon gene orcolony stimulating factor gene is feline interferon-ω gene, humaninterferon-β gene or feline granulocyte colony stimulating factor gene.11. A process for producing recombinant cytokine according to any ofclaims 1 through 3 wherein cytokine is extracted from cocoon and silkthread by using an aqueous solvent.
 12. A gene recombinant silkworm inwhich a cytokine gene has been inserted into a chromosome and cytokineis produced in silk glands or cocoon and silk thread.
 13. A generecombinant silkworm according to claim 12 wherein the cytokine geneinserted into a chromosome is an interferon gene or colony stimulatingfactor gene.
 14. A gene recombinant silkworm according to claim 13wherein the interferon gene or colony stimulating factor gene insertedinto a chromosome is feline interferon-ω gene, human interferon-β geneor feline granulocyte colony stimulating factor gene.
 15. A vector forinserting an exogenous gene into silkworm chromosomes in which acytokine gene is coupled downstream from a promoter that is specificallyexpressed in silk glands.
 16. A vector for inserting an exogenous geneinto silkworm chromosomes according to claim 15 wherein the promoter issericin gene promoter.
 17. A vector for inserting an exogenous gene intosilkworm chromosomes according to claim 15 wherein the promoter is afibroin H chain gene promoter.
 18. A vector for inserting an exogenousgene into silkworm chromosomes according to any of claims 15 through 17wherein the cytokine gene is located between a pair of inverted terminalsequences originating in a transposon.
 19. A vector for inserting anexogenous gene into silkworm chromosomes according to any of claims 15through 18 wherein the cytokine gene is an interferon gene or a colonystimulating factor gene.
 20. A vector for inserting an exogenous geneinto silkworm chromosomes according to claim 19 wherein the interferongene or colony stimulating factor gene is feline interferon-ω gene,human interferon-β gene or feline granulocyte colony stimulating factorgene.
 21. A gene cassette for expressing an exogenous protein comprising(1) a promoter expressed in silk glands, and (2) a gene coupleddownstream from (1) in which the 5′ terminal portion of fibroin H chaingene is fused to the 5′ side of an exogenous protein structural gene.22. A gene cassette for expressing an exogenous protein comprising (1) apromoter expressed in silk glands, and (2) a gene coupled downstreamfrom (1) in which the 3′ terminal portion of fibroin H chain gene isfused to the 3′ side of an exogenous protein structural gene notcontaining a stop codon, or a gene cassette for expressing an exogenousprotein comprising (1) a promoter expressed in silk glands, and (2) agene coupled downstream from (1) in which an exogenous proteinstructural gene is fused to the 3′ side of the 3′ terminal portion offibroin H chain gene.
 23. A gene cassette for expressing an exogenousprotein comprising (1) a promoter expressed in silk glands, and (2) agene coupled downstream from (1) in which the 5′ terminal portion offibroin H chain gene is fused to the 5′ side of an exogenous proteinstructural gene not containing a stop codon, and in which the 3′terminal portion of fibrin H chain gene is fused to the 3′ side of thestructural gene.
 24. A gene cassette according to claim 21 or 23 whereinthe 5′ terminal portion of the fibroin H chain gene contains a firstexon, first intron and a portion of a second exon of fibroin H chaingene.
 25. A gene cassette according to claim 24 wherein the portionwhere the first exon and second exon of the fibroin H chain gene arejoined is a secretion signal gene region of fibroin H chain gene.
 26. Agene cassette according to claim 25 wherein the promoter expressed insilk glands of (1) and the 5° terminal portion of fibroin H chain genecoupled downstream from (1) are the DNA shown in SEQ. ID No. 22 and SEQ.ID No.
 23. 27. A gene cassette according to claim 22 or 23 wherein the3′ terminal portion of the fibroin H chain gene contains at least onecodon that encodes cysteine.
 28. A gene cassette according to claim 27wherein the 3′ terminal portion of the fibroin H chain gene is the DNAshown in SEQ. ID No.
 24. 29. A gene cassette according to any of claims21 through 28 wherein the promoter expressed in silk glands is at leastone promoter selected from fibroin H chain gene promoter, fibroin Lchain gene promoter and sericin gene promoter.
 30. A gene cassetteaccording to any of claims 21 through 29 wherein at least one poly Aaddition region selected from a poly A addition region of fibroin Hchain gene, a poly A addition region of fibroin L chain gene and a polyA addition region of sericin gene is present downstream from a genecassette for expressing an exogenous protein according to any of claims21 through
 29. 31. A gene cassette for inserting a gene into chromosomesof insect cells comprising inverted repetitive sequences of a pair ofpiggyBac transposons present on both sides of a gene cassette forexpressing an exogenous protein according to any of claims 21 through30.
 32. An expression vector for insect cells that contains a genecassette for expressing an exogenous protein according to any of claims21 through
 31. 33. A gene insertion vector for insect cells thatcontains a gene cassette for inserting a gene into chromosomes of insectcells according to claim
 31. 34. A process for producing an exogenousprotein comprising inserting a vector for insect cells according toclaim 32 or 33 into insect cells.
 35. A process for producing anexogenous protein according to claim 34 wherein the insect cellsoriginate in a lepidopteron.
 36. A process for producing an exogenousprotein according to claim 35 wherein the insect cells originate insilkworm moths (Bombyx mori).
 37. A process for producing an exogenousprotein according to claim 36 wherein the insect cells are silk glandcells of silkworm moths (Bombyx mori).
 38. A process for producing anexogenous protein comprising producing a recombinant silkworm in which agene cassette for expressing an exogenous protein according to any ofclaims 21 through 31 is inserted into a chromosome using a geneinsertion vector for insect cells according to claim 33 and the DNAtransfer activity of piggyBac transposase, producing exogenous proteinin the silk glands or cocoon and silk thread of the resultingrecombinant silkworm, recovering the exogenous protein from the silkglands or silk and cocoon thread.
 39. A process for producing anexogenous protein according to claim 38 wherein the recombinantsilkworm, in which the gene cassette for expressing an exogenous proteinhas been inserted into a chromosome, is produced by simultaneouslymicro-injecting the gene insertion vector for insect cells and DNA orRNA that produces the piggyBac transposase into silkworm eggs.
 40. Arecombinant silkworm in which a gene cassette for expressing anexogenous protein according to any of claims 21 through 31 has beeninserted into a chromosome, and which has the ability to produce theexogenous protein in silk glands or silk thread.
 41. Silk threadcontaining an exogenous protein produced by a recombinant silkwormaccording to claim 40.